The non-catalytic function of PARP2 in DNA repair and cancer therapy

PARP2在DNA修复和癌症治疗中的非催化功能

• 批准号: 10540084
• 负责人: Shan Zha
• 金额: $ 59.71万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10540084
• 关键词: ADP Ribose Transferases; ADP ribosylation; Acute Myelocytic Leukemia; Address; Adult; Affect; Affinity; Anemia; Animals; BRCA deficient; BRCA1 gene; BRCA2 gene; Base Excision Repairs; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Bone Marrow; CHEK2 gene; Cancer Patient; Cells; Cellular biology; Charge; Chromatin; Chronic; Cisplatin; Clinical; Clonal Expansion; DNA; DNA Binding; DNA Damage; DNA Ligases; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA lesion; Defect; Development; Dose; Dysplasia; Embryo; Erythropoiesis; Flow Cytometry; Fluorescence; Genes; Genetic Epistasis; Goals; Hematopoiesis; Hematopoietic; Histones; Hypersensitivity; Impairment; In Vitro; Knock-in; Knockout Mice; Lasers; Ligase; Ligation; Link; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mediating; Modeling; Molecular Biology; Mus; Mutation; Myelogenous; Okazaki fragments; Patients; Photobleaching; Physical condensation; Poly Adenosine Diphosphate Ribose; Proteins; Relaxation; Risk; Role; S phase; Site; Specificity; Splenomegaly; TP53 gene; Testing; Toxic effect; Withdrawal; XRCC1 gene; base; brca gene; cancer cell; cancer therapy; chemotherapy; effective therapy; in vivo; inhibitor; live cell imaging; malignant breast neoplasm; mouse genetics; mouse model; novel; premature; prevent; recruit; repaired

PROJECT SUMMARY/ABSTRACT PARP1 and PARP2 are DNA damage-induced poly-ADP-ribose (PAR) transferases, which are recruited to and activated by DNA breaks. Active PARP1&2 PARylate themselves and histones to promote DNA repair and chromatin relaxation. Dual-specificity inhibitors (PARPi) for PARP1 and PARP2 are currently used for the treatment of BRCA1/2-deficient breast, ovarian, pancreatic, and prostate cancers. However, severe anemia occurs in ~ one-third of patients, leading to dose reduction and premature termination of PAPRi therapy. PARPi also cause significant clonal hematopoiesis, a condition that increases the risk for myeloid dysplasia and acute- myeloid leukemia (MDS/AML). These hematopoietic toxicities are unexpected since the complete loss of Parp1 that is responsible for >80% of DNA damage-induced PARylation in cells, has no impact on hematopoiesis. To understand this potential on-target toxicity of PARPi, we generated mouse models with knockin catalytically inactive mutations in Parp2 – Poly-ADP-Ribosylation (PARylation) deficient (E534A, Parp2EA) or Mono-ADP- Ribosylation (MARylation) deficient (H404A, Parp2HA). In contrast to the normal development of Parp2-/- mice, mice expressing PARylation deficient PARP2 (Parp2EA/EA) died at embryonic day 16.5 (E16.5) with severe anemia and stage-specific blocks in erythropoiesis. The Parp2HA/HA mice are viable but display splenomegaly and chronic anemia. Meanwhile, our cell biology analyses suggest that clinically used PARPi effectively stalls PARP2, but not PARP1, on laser-induced DNA damage sites. Based on these and other observations, we hypothesize that PARP2 has PARylation-dependent structural functions that preferentially block nick ligation during DNA replication, underlie the PARP inhibitors-induced erythropoiesis defects and anemia. Specifically, we propose that catalytically inactive PARP2 were trapped on DNA breaks, especially 5’pho-nicks, where they prevent other repair factors (e.g., Ligase1) from accessing the DNA nicks. Correspondingly, like the Parp2EA/EA mice, Lig1-/- mice also succumbed to lethal anemia at E16.5. To test our hypothesis, we will Aim 1) investigate the mechanism that regulates PARP2 recruitment and dynamics at DNA damage sites using live-cell imaging and bulk biochemical assays; Aim 2) determine the impacts of catalytically inactive PARP2 on the recruitment and function of other DNA repair factors, and DNA replication in vivo and in vitro; Aim 3) characterize the mouse models expressing catalytically inactive - Parp2 and investigate how the loss of Trp53, CHK2, two genes associated clonal hematopoiesis, modulates the anemia in Parp2EA/EA models. The completion of the proposal will characterize the previously unrecognized structural functions of PARP2, address how catalytically inactive PARP2 compromises DNA replication and selectively abrogates erythropoiesis, providing the strategy to mitigate this on-targeted toxicity of PARP inhibitors by reducing PARP2 stalling.

项目摘要/摘要 PARP1和PARP2是DNA损伤诱导的多聚ADP核糖(PAR)转移酶 并被DNA断裂激活。活性PARP1和PAR2自身和组蛋白促进DNA修复 染色质松弛。PARP1和PARP2的双特异性抑制剂(PARPI)目前用于 BRCA1/2缺陷乳腺癌、卵巢癌、胰腺癌和前列腺癌的治疗。然而，严重的贫血 发生在约三分之一的患者中，导致剂量减少和纸片疗法提前终止。PARPI 还会导致显著的克隆性造血，这种情况会增加骨髓发育不良和急性- 髓系白血病(MDS/AML)。由于PARP1的完全丧失，这些造血毒性是意想不到的 这是DNA损伤导致的细胞中80%的PAR化的原因，对造血没有影响。至 为了了解PARPI的这种潜在的靶向毒性，我们用催化的方法建立了敲打蛋白的小鼠模型 缺乏Parp2-聚-ADP-核糖基化(Parp2EA)或单-ADP-的非活性突变 缺乏核糖化(MAR化)(H404A，Parp2HA)。与Parp2-/-小鼠的正常发育相反， 表达PARP2(Parp2EA/Ea)的小鼠在胚胎16.5天(E16.5)死亡，患有严重的 贫血和红血球生成的阶段性阻断。Parp2HA/HA小鼠存活，但表现为脾肿大 和慢性贫血。同时，我们的细胞生物学分析表明，临床使用的PARPI有效地延缓了 PARP2，而不是PARP1，在激光诱导的DNA损伤部位。基于这些和其他观察结果，我们 假设PARP2具有依赖PAR化的结构功能，优先阻断NICK DNA复制过程中的连接，是PARP抑制剂诱导的红细胞生成缺陷和贫血的基础。 具体地说，我们认为催化失活的PARP2被捕获在DNA断裂上，特别是5‘pho-nick， 在那里它们阻止其他修复因子(例如，Ligase1)访问DNA缺口。相应地，就像 Parp2EA/EA小鼠、Lig1-/-小鼠也在E16.5死于致死性贫血。为了检验我们的假设，我们将目标定为1) 利用活细胞研究DNA损伤部位PARP2募集和动态的调控机制 成像和整体生化分析；目的2)确定催化失活的PARP2对 其他DNA修复因子的招募和功能，以及体内和体外的DNA复制；目的3)表征 小鼠模型的催化失活表达-Parp2并研究Trp53、Chk2、2的缺失 在Parp2EA/EA模型中，与克隆性造血相关的基因调控贫血。该计划的完成 提案将描述以前未被认识到的PARP2的结构功能，解决如何催化 失活的PARP2损害DNA复制并选择性地取消红细胞生成，提供了策略 通过减少PARP2停滞来减轻PARP抑制剂的这种靶向毒性。